Proteinase K is used in forensic DNA extraction methods for cell lysis and degradation of proteins. Here we compare Proteinase K with a novel protease. We conclude that there is no need to exchange Proteinase K in our methods.
DNA extraction is crucial for all downstream applications and greatly influences the chance to obtain a useful DNA profile from a trace sample. Proteinase K is widely used for cell lysis and degradation of proteins in forensic DNA extraction methods.
Here, a novel protease is investigated, referred to as Protease X. It has been developed by researchers at the Royal Institute of Technology in Stockholm, Sweden. The properties of Protease X are similar to Proteinase K with two major differences: Proteinase K has endolytic activity and an optimum at 56 °C whereas Protease X has both endolytic and exolytic activity with an optimum at 37 °C.
2. Materials and methods
Samples were prepared from human muscle tissue from one individual.
Proteinase K and Protease X were tested individually and as a mixture, and with incubation at 37 °C or 56 °C. Different incubation times, ranging from 5 min to 2 h, were tested. Five replicates and one negative control were run for each protease and incubation time.
The first sample set was purified using BioRobot® EZ1 (Qiagen) with the EZ1 DNA Investigator card and kit and with the Tip Dance protocol. The pre-treatment protocol included adding 10 μl protease (10 mg/μl) to the samples. In the mixture 5 μl of each protease was used.
The second sample set was incubated at 56 °C and purified by manual phenol–chloroform purification, followed by Centricon®-100 filtration (Millipore). 15 μl protease (10 mg/μl) were added to these samples, and 7.5 μl of each protease were used in the mixture.
Quantity and quality results were obtained using ABI 7300 Real Time PCR System with Quantifiler™ Human DNA Quantification Kit, GeneAmp® PCR System 9700 with AmpFlSTR® SGM Plus® PCR Amplification Kit, Applied Biosystems 3130xl Genetic Analyzer and GeneMapper™ID software, version 3.1 (Applied Biosystems).
Statistical analyses of the results were performed with the software Minitab® 14 (Minitab Inc.).
3. Results and discussion
DNA recovery was significantly higher for samples treated with Proteinase K at 56 °C compared to Protease X or the mixture incubated at 37 °C (Fig. 1). No statistical difference was found between the latter two. The majority of samples generated complete DNA profiles.
When testing at shorter incubation times of 5 and 10 min, phenol–chloroform extraction was used. In Fig. 2, Protease X and the mixture incubated in 56 °C are compared to Proteinase K in 56 °C, Protease X in 37 °C and the mixture in 37 °C.
The quantification results from the second sample set are summarised in Fig. 3. One sample concentration was excluded from the mean value and standard deviation calculations because of a very aberrant result. All samples generated complete DNA profiles. A two-way analysis of variance with Minitab® 14 showed that Proteinase K and the mixture of Proteinase K and Protease X obtained significantly higher DNA yields compared to Protease X, which can be due to the temperature optimum of Proteinase K. The mixture obtained slightly higher DNA concentrations than Proteinase K alone, but the difference was not significant. However, it indicates that there can be synergetic effects between Proteinase K and Protease X. The statistical analysis also showed that the time factor is negligible (data not shown).
For shorter incubation times at 56 °C, the mixture seems to be more efficient compared to Proteinase K alone. The difference is relatively small and not statistically significant in this test. We conclude that there is no need to exchange or complement Proteinase K in our DNA extraction methods.
Conflict of interest
Accepted: October 1, 2007
Received in revised form: September 10, 2007
Received: August 7, 2007
© 2008 Elsevier Ireland Ltd. Published by Elsevier Inc. All rights reserved.